Penile size in healthy Nigerian newborns: country-based reference values and international comparisons.

AIM: To establish normal reference values for penile size in Nigerian newborn boys and to compare those values with standards from other populations. METHODS: A total number of 261 healthy newborn boys delivered at gestational ages of 28 weeks or more were enrolled in the study. Penile lengths and widths were measured within 72 h of birth. RESULTS: The mean (±SD) penile length in the 261 Nigerian males studied was 3.4 ± 0.48 cm, while the mean mid-shaft diameter was 1.2 ± 0.14 cm. Compared with data from other populations, Nigerian newborn boys had similar penile sizes to those reported for US Caucasian boys (mean 3.4 cm), but significantly greater penile sizes than those reported for boys from China and Hong Kong (mean 3.0 and 3.1 cm, respectively; both p < 0.001). There was a slight, but significant, difference in size between Nigerian and Malaysian boys, with Malaysian boys having greater penile sizes (mean 3.5 cm; p < 0.05). CONCLUSION: A Nigerian newborn with a penile length of <2.39 cm can be considered to have a micropenis.

Expression of beta-nerve growth factor and its receptor in rat seminiferous epithelium: specific function at the onset of meiosis.

beta-Nerve growth factor (NGF) is expressed in spermatogenic cells and has testosterone-downregulated low-affinity receptors on Sertoli cells suggesting a paracrine role in the regulation of spermatogenesis. An analysis of the stage-specific expression of NGF and its low affinity receptor during the cycle of the seminiferous epithelium in the rat revealed NGF mRNA and protein at all stages of the cycle. Tyrosine kinase receptor (trk) mRNA encoding an essential component of the high-affinity NGF receptor was also present at all stages. In contrast, expression of low affinity NGF receptor mRNA was only found in stages VIIcd and VIII of the cycle, the sites of onset of meiosis. The low-affinity NGF receptor protein was present in the plasma membrane of the apical Sertoli cell processes as well as in the basal plasma membrane of these cells at stages VIIcd to XI. NGF was shown to stimulate in vitro DNA synthesis of seminiferous tubule segments with preleptotene spermatocytes at the onset of meiosis while other segments remained nonresponsive. We conclude that NGF is a meiotic growth factor that acts through Sertoli cells.

Endocrine dysfunction in Prader-Willi syndrome: a review with special reference to GH.

Prader-Willi syndrome is a genetic disorder occurring in 1 in 10,000-16,000 live-born infants. In the general population, approximately 60 people in every 1,000,000 are affected. The condition is characterized by short stature, low lean body mass, muscular hypotonia, mental retardation, behavioral abnormalities, dysmorphic features, and excessive appetite with progressive obesity. Furthermore, morbidity and mortality are high, probably as a result of gross obesity. Most patients have reduced GH secretory capacity and hypogonadotropic hypogonadism, suggesting hypothalamic-pituitary dysfunction. Replacement of GH and/or sex hormones may therefore be beneficial in Prader-Willi syndrome, and several clinical trials have now evaluated GH replacement therapy in affected children. Results of GH treatment have been encouraging: improved growth, increased lean body mass, and reduced fat mass. There was also some evidence of improvements in respiratory function and physical activity. The long-term benefits of GH treatment are, however, still to be established. Similarly, the role of sex hormone replacement therapy needs to be clarified as few data exist on its efficacy and potential benefits. In summary, Prader-Willi syndrome is a disabling condition associated with GH deficiency and hypogonadism. More active treatment of these endocrine disorders is likely to benefit affected individuals.

Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility.

Locally produced estrogen promotes fetal rat metatarsal bone growth; an effect mediated through increased chondrocyte proliferation and decreased apoptosis.

The importance of estrogens for the regulation of longitudinal bone growth is unequivocal. However, any local effect of estrogens in growth plate cartilage has been debated. Recently, several enzymes essential for estrogen synthesis were shown to be expressed in rat growth plate chondrocytes. Local production of 17beta-estradiol (E2) has also been demonstrated in rat costal chondrocytes. We aimed to determine the functional role of locally produced estrogen in growth plate cartilage. The human chondrocyte-like cell line HCS-2/8 was used to study estrogen effects on cell proliferation (3H-labeled thymidine uptake) and apoptosis (cell death detection ELISA kit). Chondrocyte production of E2 was measured by RIA and organ cultures of fetal rat metatarsal bones were used to study the effects of estrogen on longitudinal growth rate. We found that significant amounts of E2 were produced by HCS-2/8 chondrocytes (64.1 +/- 5.3 fmol/3 days/10(6) cells). The aromatase inhibitor letrozole (1 microM) and the pure estrogen receptor antagonist ICI 182,780 (10 microM) inhibited proliferation of HCS-2/8 chondrocytes by 20% (P < 0.01) and almost 50% (P < 0.001), respectively. Treatment with ICI 182,780 (10 microM) increased apoptosis by 228% (P < 0.05). Co-treatment with either caspase-3 or pan-caspase inhibitors completely blocked ICI 182,780-induced apoptosis (P < 0.001 vs ICI 182,780 only). Moreover, both ICI 182,780 (10 microM) and letrozole (1 microM) decreased longitudinal growth of fetal rat metatarsal bones after 7 days of culture (P < 0.01). In conclusion, our data clearly show that chondrocytes endogenously produce E2 and that locally produced estrogen stimulates chondrocyte proliferation and protects from spontaneous apoptosis. In addition, longitudinal growth is promoted by estrogens locally produced within the epiphyseal growth plate.

In vitro stimulation of stage-specific deoxyribonucleic acid synthesis in rat seminiferous tubule segments by interleukin-1 alpha.

Levels of rat testicular interleukin-1-like factor (tIL-1) have been shown to correlate with DNA synthetic activity during the cycle of the rat seminiferous epithelium, suggesting its role as a spermatogonial or meiotic growth factor. To explore this further, a new in vitro model system was developed. Rat seminiferous tubule segments from stages I, V, VIIa, and VIII-IX of the cycle were isolated by transillumination-assisted microdissection, cultured in chemically defined serum-free medium supplemented with human recombinant IL-1 alpha, and labeled with [3H]thymidine. During incubation, spontaneous progression of spermatogenesis was noted. Inactive stage VIIa tubule segments differentiated to stage VIII and initiated DNA synthesis, and concomitantly started to secrete IL-1-like factor. DNA synthesis of stages VIII-IX ceased through differentiation of spermatocytes to leptotene-zygotene (stages XII-XIII of the cycle). IL-1 alpha stimulated DNA synthesis significantly in spermatogonia of stage I. Meiotic DNA synthesis at stage VIIa was stimulated (48 h/34 C) and maintained at stages VIII-IX (48 h/34 C). IL-1 alpha seems to act as a regulator of spermatogenic DNA synthesis in both mitotic and meiotic phases. It has mainly stimulating and maintaining effects, but it may also be inhibitory under certain conditions.

FSH stimulation of testicular androgen binding protein (ABP): comparison of ABP response and ovarian augmentation.

Production of testicular androgen binding protein (ABP), ceases following hypophysectomy and can be stimulated by FSH. Within 24 h after the administration of FSH, ABP can be measured in caput epididymis supernatant and by 4 days after FSH treatment, the concentration of ABP reaches a plateau. In a 3-day assay, ABP production in immature hypophysectomized rats was stimulated by 31 mug NIH-FSH-P1 per day (0.08 U NIH-FSH-P1 per 3 days) which is comparable to the sensitivity of the ovarian weight augmentation test in hypophysectomized rats. The relative ovarian weight augmenting and ABP stimulating activities of various FSH preparations were in agreement, suggesting that the biological stimulus of the ABP response is, in fact, FSH. The ABP response to FSH could become a useful testicular bioassay for FSH. Such an assay would be more practicle if ABP could be measured by a radioimmunoassay.

21-hydroxylase deficiency: disease-causing mutations categorized by densitometry of 21-hydroxylase-specific deoxyribonucleic acid fragments.

The types of disease-causing mutations were studied in 43 unrelated patients with 21-hydroxylase deficiency. Densitometry of Southern blots after cleavage with the restriction enzymes TaqI, PvuII, and BglII was used to measure the ratio of the copy-number of the 21-hydroxylase gene (CYP21) to the copy-number of its pseudogene (CYP21P). DNA from 16 unrelated patients showed equal hybridization intensities of the 2 genes, indicating that point mutations caused the enzyme deficiency. One of the 2 haplotypes in 7 patients showed evidence of a large gene conversion between the CYP21 and the CYP21P gene without loss of the total number of 21-hydroxylase genes. Deletion of at least 1 21-hydroxylase gene was found in 11 patients. DNA from 8 of these patients had relative hybridization intensities compatible with a deletion of the active 21-hydroxylase gene, CYP21. Two patients with the salt-wasting form of the disease showed homozygous loss of DNA fragments that are specific for the 5' end of the active 21-hydroxylase gene. Nine patients showed relative 21-hydroxylase hybridization intensities compatible with duplication of the gene in 1 or both haplotypes. In conclusion, point mutations, gene conversions, or CYP21 gene deletions are the typical mutations in patients with the simple virilizing and salt-wasting forms of the disease, while duplications of the locus are overrepresented in patients with nonclassical 21-hydroxylase deficiency.

Subnormal androgen and elevated progesterone levels in women treated for congenital virilizing 21-hydroxylase deficiency.

Clinical, anthropometric, and endocrine data were examined in 22 corticosteroid-treated, prenatally virilized women with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency and in 22 matched healthy controls. In view of the androgen excess, limited growth, and subfertility associated with CAH, the investigation focused on androgenic/anabolic status and circulating progesterone levels. CAH patients were shorter and had a significantly higher body mass index than the controls. One pregnancy was reported in the CAH group compared to 15 in the controls. Five of the CAH patients were judged as undersubstituted based on greatly elevated circulating levels of 17 alpha-hydroxyprogesterone. These five patients had elevated serum levels of progesterone (P) and testosterone (T) and elevated ratios between T and sex hormone-binding globulin, but subnormal levels of dehydroepiandrosterone (DHA) and its sulfate. The remaining 17 well substituted patients had elevated follicular phase levels of P, but subnormal levels of all androgens (4-androstene-3,17-dione, T, DHA, and DHA sulfate) and subnormal T/sex hormone-binding globulin ratios. Contrary to the apprehension that normally guides the treatment of CAH, well substituted patients may be considered hypoandrogenic rather than hyperandrogenic. The elevated levels of P may have a minipill-like effect, which may be one of the causes of the differences in fertility between salt-wasting and simple virilizing CAH.

Failure of cortisone acetate treatment in congenital adrenal hyperplasia because of defective 11beta-hydroxysteroid dehydrogenase reductase activity.

Congenital adrenal hyperplasia in children is often treated with cortisone acetate and fludrocortisone. It is known that certain patients with congenital adrenal hyperplasia require very high substitution doses of cortisone acetate, and a few patients do not respond to this treatment at all. A patient with 21-hydroxylase deficiency, for whom elevated pregnanetriol (P3) levels in urine were not suppressed during treatment with cortisone acetate (65 mg/m2 x day), was examined. The activation of cortisone to cortisol was assessed by measuring urinary metabolites of cortisone and cortisol. The patient's inability to respond to treatment with cortisone acetate was found to be caused by a low conversion of cortisone to cortisol, assumed to be secondary to low 11beta-hydroxysteroid dehydrogenase activity (11-oxoreductase deficiency). All exons and exon/intron junctions of the 11beta-hydroxysteroid dehydrogenase type1 gene (HSD11L) were sequenced without finding any mutations, but a genetic lesion in the promoter or other regulatory regions cannot be ruled out. The deficient 11-oxoreductase activity seems to have been congenital, in this case, but can possibly be attributable to a down-regulation of the enzyme activity. The results support the use of hydrocortisone, rather than cortisone acetate, for substitution therapy in adrenal insufficiency.

Mutational spectrum of the steroid 21-hydroxylase gene in Sweden: implications for genetic diagnosis and association with disease manifestation.

We have characterized the disease-causing mutations in the steroid 21-hydroxylase genes of 127 patients with different clinical forms of congenital adrenal hyperplasia, representing 186 unrelated chromosomes. The gene was completely absent on 29.8% of the chromosomes, and this together with the I2 splice (27.7%), I173N (20.8%), V282L (5.4%), and R357W (3.8%) mutations constitute 87.5% of all affected chromosomes. In total, 15 different sequence aberrations combine to form 19 different disease-causing alleles. The results confirm that genotyping is an efficient means of diagnosing steroid 21-hydroxylase deficiency, although special consideration is needed to resolve genotypes when full families are not available. Clinical presentations of the different combinations of mutations indicate that genotyping is reliable for prediction of clinical outcome in patients with 21-hydroxylase deficiency. It is especially helpful in determining whether in utero treatment of affected females is indicated and in classifying the severity of 21-hydroxylase deficiency in children diagnosed through neonatal screening, before symptoms have appeared.

Prophylactic adrenalectomy of a three-year-old girl with congenital adrenal hyperplasia: pre- and postoperative studies.

Long term follow-up studies of children with congenital adrenal hyperplasia have documented less than desirable outcomes, including reduction in final adult height, obesity, virilism, and decreased fertility. We have proposed that children with the most severe forms of congenital adrenal hyperplasia would be better off if their adrenals were removed at an early age. We report here on our experience with prophylactic bilateral adrenalectomy in a 3-yr-old girl with a double null mutation of the CYP21 gene. The results of sodium balance studies, performed preoperatively on our patient and her unaffected fraternal twin sister, and hormonal data are presented as well. In contrast to her twin, who markedly increased her sodium retention in response to ACTH, our patient showed increased natriuresis, suggesting a deleterious effect of her adrenals on sodium homeostasis. Adrenalectomy was carried out at the time of necessary genital repair. No surgical or postsurgical complications were encountered.

Long-term somatic follow-up of prenatally treated children with congenital adrenal hyperplasia.

Prenatal virilization of female fetuses is a serious symptom associated with severe congenital adrenal hyperplasia. In attempt to avoid sexual ambiguity, prenatal treatment of 21-hydroxylase deficiency was initiated in 1984, with the first Scandinavian case treated in 1985. Here we have studied the outcome of prenatal diagnosis and therapy of 44 at-risk pregnancies monitored during the years 1985-1995 in Scandinavia. Treated mothers and children were compared with matched controls. Compared to their elder affected sisters, all 5 girls with severe congenital adrenal hyperplasia who were treated until term showed little virilization. Only 1 required surgery for labial fusion. The majority of the 44 dexamethasone-treated fetuses demonstrated normal pre- and postnatal growth compared to matched controls. However, several adverse events such as failure to thrive and delayed psychomotor development, were reported among the treated infants. In addition, treated mothers reported more side-effects during pregnancy than did controls. A significant increase in weight gain was observed during early pregnancy when treatment was initiated, but this initial rapid weight gain declined during late pregnancy or when treatment was terminated. Thus, experience to date suggests that prenatal treatment of affected female fetuses is generally efficient in minimizing virilization of external genitalia. However, there is still a need to collect more data concerning possible rare unfavorable effects of this therapy on mother and child.

Demonstration of estrogen receptor-beta immunoreactivity in human growth plate cartilage.

Estrogens affect longitudinal bone growth through their action on endochondral bone formation. Two estrogen receptors are known, the classical estrogen receptor-alpha (ER alpha), newly demonstrated in human growth plate cartilage, and a recently cloned estrogen receptor-beta (ER beta). The present study aimed to localize a possible expression of ER beta protein in human growth plates. Tissue samples were obtained from tibial and femoral growth plates in four female pubertal patients undergoing epiphyseal surgery. Immunohistochemistry, using two different ER beta-specific antibodies, demonstrated positive staining for ER beta in hypertrophic epiphyseal chondrocytes from all patients. No staining was noted in resting or proliferative chondrocytes. These data suggest that in addition to ER alpha, human epiphyseal chondrocytes also express ER beta. The physiological role of ER beta in the regulation of longitudinal bone growth in humans remains to be elucidated.

Identification of the main gonadal autoantigens in patients with adrenal insufficiency and associated ovarian failure.

Autoimmune adrenal insufficiency is associated with premature ovarian failure at frequencies of 10-20%. In these patients a reactivity against an unknown steroid cell antigen in both the adrenal glands and gonads has been described. We have recently identified the cytochrome P450 enzyme 21-hydroxylase and the side-chain cleavage enzyme (SCC) as the major adrenal autoantigens in Addison's disease and the rare autoimmune polyendocrine syndrome type I (APS-I), respectively. In an attempt to identify the steroid cell antigen, sera from patients with Addison's disease (n = 13) and APS-I (n = 7) that reacted with Leydig cells were selected. Preparations of isolated human granulosa cells, isolated rat Leydig cells, and fractions of human placenta were used in Western blots. All sera were also tested against bacterially expressed 21-hydroxylase, SCC, and 17 alpha-hydroxylase. The SCC was recognized by sera from patients with Addison's disease and those with APS-I. In addition, a majority of the sera (n = 12) reacted with a novel 51-kilodalton autoantigen present in granulosa cells and placenta. The results show that the steroid cell autoantigen consists mainly of the SCC and a novel protein of unknown function.

Normal lateralization for handedness and ear advantage in a verbal dichotic listening task in women with congenital adrenal hyperplasia (CAH).

Functional cerebral asymmetry was studied in 22 women, 17-34 years old, with prenatal virilization due to congenital adrenal hyperplasia (CAH) (21-hydroxylase deficiency) and 22 matched, healthy controls. The theory of an androgen influence on cerebral lateralization has been supported by the observation of sex differences in cognitive performance, anatomical cerebral differences between the sexes and atypical patterns of functional asymmetry in certain anomalous hormonal states. The result of the present study did however not show a significantly different pattern of functional asymmetry for women with CAH and prenatal exposure to high levels of androgens. No significant differences with the controls were found for the Finger Tapping Test (FTT), Edinburgh Handedness Inventory (EHI) or Dichotic Listening (Consonant-Vowel) Test (DL). The CAH group included four non-right-handers (18%) vs two (9%) in the control group. As sex differences generally are small, the present result, may have been influenced by the limited size of the sample.

Estrogen treatment of tall girls: risk of thrombosis?

In 34 girls who were treated with large doses of ethinylestradiol because of expected excessive tall stature, antithrombin activity in the blood was followed before, during, and after treatment. All girls showed lower levels of antithrombin during treatment than before or after estrogen administration; the mean concentration during treatment was at or below the lower limit of control values (-2 SD). In three girls, the antithrombin levels were so low that the medication was withdrawn. There was no difference between the mean levels of antithrombin activity in the three groups treated with 0.25, 0.50, or 1.0 mg of ethinylestradiol. Deficiency of antithrombin is known to be associated with an increased risk of thrombosis. Therefore, antithrombin concentrations in blood should be determined before and during treatment with large doses of estrogens.

Growth retardation induced by dexamethasone is associated with increased apoptosis of the growth plate chondrocytes.

Glucocorticoids cause significant growth retardation in mammals and humans and decreased proliferation of chondrocytes has been considered as the main local mechanism. Death by apoptosis is an important regulator of homeostasis in multicellular organisms. Here we chose to study the role of apoptosis in growth retardation caused by glucocorticoid treatment. We treated 7-week-old male rats with dexamethasone (5 mg/kg/day) for 7 days. Apoptosis was studied in tibiae growth plates by the TUNEL method. Immunoreactivity for parathyroid hormone-related peptide (PTHrP), caspase-3, and the anti-apoptotic proteins Bcl-2 and Bcl-x was also studied. Apoptosis was mainly localized in terminal hypertropic chondrocytes (THCs) in both control and dexamethasone-treated animals. Dexamethasone caused an increase in apoptosis which was fourfold in THCs (2.45+/-0.12 vs 0.62+/-0.09 apoptotic cells/mm growth plate, P<0.001), and 18-fold in proliferative chondrocytes (0.18+/-0.04 vs 0.01+/-0.007 apoptotic cells/mm growth plate, P<0.001). Increased apoptosis after dexamethasone treatment was accompanied by increased immunoreactivity for caspase-3 and decreased immunoreactivity for the anti-apoptotic proteins Bcl-2 and Bcl-x, which further supports our apoptosis results. Dexamethasone also decreased the immunoreactivity for PTHrP, suggesting a role in the mechanism by which glucocorticoids induce apoptosis in the growth plate. We conclude that apoptosis is one mechanism involved in growth retardation induced by glucocorticoids. Premature loss of resting/proliferative chondrocytes by apoptosis could contribute to incomplete catch-up seen after prolonged glucocorticoid treatment.

Effects of hysterectomy and in-vivo treatment with uterine extracts on plasma concentrations of growth hormone, thyrotrophin and thyroid hormones in rats: a kinetic study.

Plasma concentrations of GH, TSH, tri-iodothyronine (T3) and thyroxine (T4) were measured in adult rats 2 and 4 weeks after ovariectomy and and ovariohysterectomy. Two weeks after ovariohysterectomy, the concentration of GH was significantly higher, but TSH and T3 concentrations were significantly lower than in rats which had been ovariectomized only. Hysterectomy had no effect on plasma GH and TSH concentrations if it was performed 2 weeks after ovariectomy. Plasma T3 had decreased by 2 weeks after ovariectomy but returned to pretreatment levels by 4 weeks. Recovery of the plasma T3 concentration was not observed if ovariectomy was followed by hysterectomy, since a further decrease of plasma T3 occurred. Plasma T4 was not significantly influenced either by ovariectomy or by ovariohysterectomy. Steroid-free uterine extracts given i.p. to ovariohysterectomized rats reduced plasma GH within 24 h of injection. Increases in plasma TSH, T3 and T4 were achieved in ovariohysterectomized rats with injections of uterine extracts (from intact, oestrogen-treated or castrated rats), but the increases were not consistent for the three hormones either as regards time after injection, nor for which particular extracts were effective. It concluded that the uterus may contain factors which influence the GH storage and secretion and TSH-thyroid regulation in rats.

Plasma growth hormone pattern and androgens influence the levels of corticosteroid-binding globulin in rat serum.

The serum concentration of corticosteroid-binding globulin (CBG) is higher in female rats than in males. Combined hypophysectomy and gonadectomy of female rats reduced the serum concentration of CBG as measured by steady-state polyacrylamide gel electrophoresis, whereas hypophysectomy of male rats increased serum CBG. These effects were seen despite replacement therapy with thyroxine and glucocorticoids. Moreover, neither androgen nor oestrogen treatment affected the serum concentrations of CBG in hypophysectomized rats. Continuous infusions of human or bovine GH (1.4 U/kg per day), by means of osmotic minipumps for 1 week, increased serum concentrations of CBG in both hypophysectomized male and female rats. In contrast, intermittent GH replacement therapy by s.c. injections at 12-h intervals either had no effect or suppressed serum CBG levels. In male rats, neonatal (days 1-2) gonadectomy increased CBG levels more than did prepubertal (day 25) gonadectomy, and testosterone replacement therapy reversed these effects. It is concluded that GH increases the serum CBG levels of hypophysectomized rats when it is given in a continuous manner, but not when given intermittently. The sex difference in serum CBG levels of normal rats may, therefore, be attributed to the more continuous secretory pattern of GH previously observed in female rats.